Extracellular Concentration of _L-Cystine Determines the Sensitivity to System x_c^- Inhibitors

Abstract

Targeting the cystine/glutamate exchange transporter, system xc -, is a promising anticancer strategy that induces ferroptosis, which is a distinct form of cell death mediated by iron-dependent lipid peroxidation. The concentration of L-cystine in culture medium is higher than the physiological level. This study was aimed to evaluate the effects of L-cystine concentration on the efficacy of ferroptosis inducers in hepatocellular carcinoma cells. This study showed that treatment with sulfasalazine or erastin, a system xcinhibitor, decreased the viability of Huh6 and Huh7 cells in a dose-dependent manner, and the degree of growth inhibition was greater in medium containing a physiological L-cystine concentration of 83 µM than in commercial medium with a concentration of 200 µM L-cystine. However, RSL3, a glutathione peroxidase 4 inhibitor, decreased cell viability to a similar extent in media containing both L-cystine concentrations. Sulfasalazine and erastin significantly increased the percentages of propidium iodide-positive cells in media with 83 µM L-cystine, but not in media with 200 µM L-cystine. Sulfasalazine-or erastin-induced accumulation of lipid peroxidation as monitored by C11-BODIPY probe was higher in media with 83 µM L-cystine than in media with 200 µM L-cystine. In contrast, the changes in the percentages of propidium iodide-positive cells and lipid peroxidation by RSL3 were similar in both media. These results showed that sulfasalazine and erastin, but not RSL3, were efficacious under conditions of physiological Lcystine concentration, suggesting that medium conditions would be crucial for the design of a bioassay for system xcinhibitors.